The botulinum neurotoxins (BoNT) are a large protein toxin family grouped into seven BoNT serotypes (A-G) based upon limited cross protection of ?-sera against each BoNT serotype. BoNT are the most toxic proteins known for humans and the causative agent of botulism. Currently, there is no licensed vaccine against botulism and the experimental penta-serotype toxoid vaccine previously available from the CDC for at-risk populations was discontinued in 2011. Thus, there is a need to develop a potent and effective BoNT vaccine against all BoNT serotypes to protect at-risk humans from exposure, including civilians in harm?s way, first responders, the military, and researchers. BoNT are organized into three domains involved in catalysis (LC), LC translocation (HCN), and receptor binding (HCC). Earlier efforts have focused on developing recombinant HCC-based vaccines to overcome the shortcomings of chemically inactivated toxoids, but recent studies have shown that recombinant full-length BoNT vaccines are more potent than vaccines comprising the receptor binding domain. In addition, ELISA studies implicated the HCN translocation domain as the immunodominant domain, not the HCC receptor binding domain, in recombinant full-length BoNT vaccinated mice surviving native botulinum toxin challenge. This supports the hypothesis that a recombinant full-length non-toxic BoNT mutated to inactivate the three independent functions of toxin action (catalysis, LC translocation, and receptor binding) will improve vaccine potency for outbred populations. The current study will utilize informatics and assessment of structure-function alignments of the seven serotypes of botulinum toxin, along with cell biological analysis and immunological assessment of the antibody (IgM and IgG) response of animals immunized with recombinant, full-length BoNT vaccine versus chemically inactivated botulinum toxoid. Two models for botulinum toxin vaccines will be tested: a single high dose BoNT vaccine for rapid response to threats of BoNT exposure and a low dose BoNT vaccine for long term protection against BoNT exposure. The low dose protective vaccine will be tested versus chemically inactivated botulinum toxoid in mice and rabbits. Understanding of the structure-function properties of bacterial toxins allows production of Next Generation vaccines that are safer, less expensive, easier to produce, and genetically malleable for rapid modification than chemically inactivated toxoids. The studies proposed in this application provide future directions for these advances in toxin vaccinology.